PROJECT SUMMARY Fanconi anemia (FA) is caused by mutations in one of at least 19 FA complementation (FANC) group genes, which are required to repair DNA interstrand cross links (ICL). Patients with FA have congenital anomalies, hematopoietic stem cell (HSC) injury resulting in increased susceptibility to bone marrow failure (severe aplastic anemia: SAA) and leukemia, increased sensitivity to chemotherapy and radiation, and high risk of secondary cancers, even if they are successfully treated by HSC transplantation for their aplastic anemia or leukemia. Because the mutations underlying FA affect every cell in the body, there is currently no cure for FA. Recent studies have demonstrated that ICL are caused by reactive aldehydes, such as acetaldehyde (MeCHO). Oxidation by the aldehyde dehydrogenase (ALDH) family of enzymes detoxify aldehydes. In East Asia, a highly prevalent point mutation in the Aldehyde Dehydrogenase 2 (ALDH2) gene causes a semidominant loss of function, and decreased ability to oxidize highly reactive MeCHO. Exposure to MeCHO occurs through generation by endogenous pathways, exogenous ingestion or inhalation, or as the major product of ethanol (EtOH) metabolism. The ALDH2*2 mutation results in the well-known ?Asian flushing syndrome? marked by a disulfiram-like response to ingestion of small amounts of EtOH. The ALDH2*2 mutation also results in increased susceptibility to cancer, especially esophageal cancer. Thus, the ALDH2 and FA DNA repair pathways confer two tiers of genome protection from the toxic effects of MeCHO. Humans and mice doubly mutated for both pathways rapidly develop bone marrow failure due to the loss of HSC. Our analyses of mice with a knock-in of the human ALDH2*2 mutation at the murine ALDH2 allele show that, even in a basal environment, (without exposure to EtOH) HSC are 1) progressively lost, 2) at a competitive disadvantage to normal HSC, and 3) have a gene expression profile typical of a response to interferons, which are known inhibitors of stem cell self-renewal. Preliminary results also indicate that EtOH exposure causes significant decline in HSC numbers in wildtype (ALDH2*1/*1) mice. We have developed ALDH activators (Aldas), small molecules which increase the activity of both ALDH2*1 and ALDH2*2, or re-direct other ALDH family members, e.g., ALDH3a1, to also oxidize MeCHO. To measure the cellular aldehydic load, we have also made unique fluorescence based sensors, which can be used in flow cytometric analyses of individual cells, e.g., HSC. In this grant we will test the hypothesis that Aldas can protect FA HSC from genotoxic injury by decreasing the load of reactive aldehydes. We will study murine models doubly mutated for FANCD2 and ALDH2*2 to determine the protective effect of ALDH2 activation, measure the effects of Aldas on aldehydic load in HSC, and determine whether recruitment of ALDH3a1 to MeCHO metabolism protects HSC. These pre-clinical studies have the potential to rapidly translate to much needed clinical trials aimed at prevention of SAA, leukemia and cancer in patients with FA.